Trophic ecology of Angolan cold-water coral reefs (SE Atlantic) based on stable isotope analyses

Cold-water coral (CWC) reefs of the Angolan margin (SE Atlantic) are dominated by Desmophyllum pertusum and support a diverse community of associated fauna, despite hypoxic conditions. In this study, we use carbon and nitrogen stable isotope analyses (δ13C and δ15N) to decipher the trophic network of this relatively unknown CWC province. Although fresh phytodetritus is available to the reef, δ15N signatures indicate that CWCs (12.90 ± 1.00 ‰) sit two trophic levels above Suspended Particulate Organic Matter (SPOM) (4.23 ± 1.64 ‰) suggesting that CWCs are highly reliant on an intermediate food source, which may be zooplankton. Echinoderms and the polychaete Eunice norvegica occupy the same trophic guild, with high δ13C signatures (-14.00 ± 1.08 ‰) pointing to a predatory feeding behavior on CWCs and sponges, although detrital feeding on 13C enriched particles might also be important for this group. Sponges presented the highest δ15N values (20.20 ± 1.87 ‰), which could be due to the role of the sponge holobiont and bacterial food in driving intense nitrogen cycling processes in sponges’ tissue, helping to cope with the hypoxic conditions of the reef. Our study provides first insights to understand trophic interactions of CWC reefs under low-oxygen conditions.


Results
Carbon and nitrogen stable isotope (δ 13 C and δ 15 N) analyses were conducted on benthic megafauna samples and on three types of Particulate Organic Matter (POM; filtered Suspended Particulate Organic Matter (SPOM), SPOM from a sediment trap and POM from sediment) collected from the Angolan CWC reefs. The lowest measured δ 13 C value was for SPOM sampled at 342 m water depth (− 22.46 ± 0.34 ‰) and the highest was for Echinus sp. (− 12.81 ± 1.65 ‰), while δ 15 N values ranged from 2.00 ± 1.48 ‰ for SPOM sampled at 532 m water depth to 21.90 ‰ in a Hexactinellid sponge. The carbon and nitrogen stable isotope bi-plot ( Fig. 1) represents a total isotopic range of 9.65 ‰ for δ 13 C and of 19.9 ‰ for δ 15 N, with a correlation of R = 0.74 (p-value < 2.2 × 10 -16 ) between δ 13 C and δ 15 N. The four clusters obtained with the k-means analysis match with the trophic position (TP) calculations (Table 1), indicating that each cluster represents a different trophic guild in the Angolan CWC reefs.
The three types of POM analyzed are included in cluster 1, presenting a mean stable isotopic composition of − 21.57 ± 0.59 ‰ and 4.83 ± 1.58 ‰ for δ 13 C and δ 15 N, respectively. Within this cluster, sediment sampled at 345 m water depth presented the most enriched values (− 20.84 ‰ for δ 13 C and 6.19 ‰ for δ 15 N), being 4.19 ‰ more enriched, in terms of δ 15 N, than SPOM sampled at 532 m water depth. The two types of SPOM analyzed (filtered SPOM and SPOM from the trap), presented a mean δ 13  The stable isotopic signature of consumers ranged from 10.52 ± 1.07 ‰ to 21.9 ‰ in δ 15 N, corresponding to M. oculata and a hexactinellid sponge, respectively, whereas the consumer with most the δ 13 C-depleted signature was D. pertusum (− 19.77 ± 1.0 ‰) and with the most δ 13 C-enriched signature was Echinus sp. (− 12.81 ± 1.65 ‰).
The consumers included in cluster 2 varied from − 19.77 ± 1.0 ‰ to − 17.25 ‰, in terms of δ 13 C and from 10.52 ± 1.07 ‰ to 14.00 ‰, in terms of δ 15 N, presenting a mean δ 13 C value of − 18.70 ± 0.83 ‰ and a mean δ 15 N value of 12.90 ± 1.00 ‰. The mean calculated TP of this cluster is 3.54 ± 0.29, with the maximum TP (3.87) occupied by a polynoid annelid (Polynoidae sp.). The consumers included in this cluster are all analyzed CWC species as well as the above-mentioned annelid (Polynoidae sp.), a hydrozoan (unidentified), a sea anemone (Actinaria) and a fish (Myctophidae sp.). The distance between the mean stable isotope ratios of cluster 1 (POM) to cluster 2 is 2.9 ‰ for δ 13 C and 8.07 ‰ for δ 15 N, which corresponds to 2.37 trophic levels, if a trophic enrichment for δ 15 N of 3.4 ‰ is considered.
All the echinoderm taxa analyzed, plus the polychaete Eunice norvegica are part of cluster 3. This group presents the most enriched carbon stable isotope signatures with mean δ 13 C of − 14.00 ± 1.08 ‰ and mean δ 15 N of 15.00 ± 1.59 ‰. The mean TP of this group is 4.16 ± 0.47.
The organism most enriched in δ 15 N was a glass sponge (Hexactinellida sp.; 21.90 ‰). All analyzed sponge taxa (cluster 4) presented the highest mean δ 15 N ratios (mean 20.20 ± 1.87 ‰) consequently corresponding, according to the TP calculations, to the highest mean TP in the trophic web (mean TP of 5.69 ± 0.53).

Discussion
The basic structure of the food web of CWC reefs offshore Angola was deciphered, for the first time. Our results indicate a reef trophic web composed of four different trophic guilds (Fig. 2). The carbon isotope signatures of POM (− 22.46 to − 20.84 ‰) indicate that the CWC reefs are sustained by photosynthesis-derived primary production from the photic zone, consistent with what was first shown by Le Guilloux et al. 78 ; nonetheless, the gap of one trophic level between POM (cluster 1) and suspension feeders (cluster 2) suggests that an additional food source was missed in our sampling. Although the significant correlation between δ 13 C and δ 15 N indicates a network supported by a single primary source 65,79,80 , the enriched δ 13 C signatures of predators and detritusfeeders (− 15.39 to − 12.81 ‰) and the high δ 15 N values of sponges (17.33 to 21.90 ‰) point to different food sources for the consumers at the Angolan CWC reefs.
The Angolan basin is an area with exceptionally high primary productivity, derived from the Benguela upwelling system 46,81 , and fueled by nutrients delivered by the Cuanza and Congo Rivers 82 . It has been argued that a nutritive food source delivered by the upwelling system could play an important role in allowing the corals to tolerate the potential stress that hypoxia and high temperatures off Angola could cause 25,26,83,84 , since an abundant and easily digestible food source could help balance the energetic metabolic demands of species under oxygen stress 47,48 . Indeed, Hanz et al. 11 , showed that high-quality and abundant OM, in the form of fresh phytodetritus, is available to the CWC reefs, corroborated by the low isotopic signature of δ 15 N of SPOM, as well as the low C/N ratios and the little degraded phytopigments of the analyzed OM 11 . In addition, fluvial input Bi-plot of the mean carbon (δ 13 C) and nitrogen (δ 15 N) stable isotopes of the main benthic megafauna groups on the Angola CWC reefs and the analyzed particulate organic matter, sampled at different depths: suspended particulate organic matter (SPOM), settling SPOM collected with a sediment trap (SPOMtrap) and sediment. Dashed grey lines represent standard deviation. Polygons represent k-means clusters, with the optimal number of clusters decided based on the elbow, silhouette and gap statistic methods. Labels in the bi-plot represent the abbreviations of the different taxonomic groups, found in  11 . Our results show that D. pertusum and M. oculata are 2 ‰ enriched compared to the mean δ 13 C signature of the analyzed POM (see cluster 1, Fig. 1), indicating that possibly the corals do not directly feed on the terrigenousenriched POM. However, the high input of nutrients associated with the terrestrial material transported with the riverine discharges could, instead, be important to maintain the primary productivity that supports the baseline of the trophic web 85 . The OM fluxes to the Angolan basin fluctuate seasonally 86 , because of seasonal changes in the upwelling system and riverine discharges 87 . It is possible that this seasonality is also reflected in the diet of the CWCs, with the corals exploiting fresh phytodetritus, when available, but also opportunistically feeding on other types of food, when fresher phytodetritus is absent 1,45,88 . At the Angolan CWC reefs, D. pertusum and M. oculata presented slightly higher δ 15 N values (11.56 ± 1.37 ‰ and 10.52 ± 1.07 ‰, respectively) than the ones reported for other CWC reefs (7.6 to 10.1 ‰ for D. pertusum and 6.9 to 10 ‰ for M. oculata) in the NE Atlantic Ocean 34,35,37,89 and the Mediterranean Sea 31 , even though the δ 15 N signature of SPOM in our study (2.00 to 5.32 ‰) is comparable to the ones reported in literature (2.2 to 8.3 ‰ 31,34,35,37,41,44,79 ). In our study area in the SE Atlantic Ocean, δ 15 N ratios show an enrichment of 7.33 ‰ and 6.29 ‰ between SPOM and D. pertusum and M. oculata, respectively. In other studies, the observed δ 15 N enrichment between D. pertusum and SPOM ranged from 2.7 to 7.1 ‰ 31,34,35,37,44 . The observed isotopic enrichment at the Angolan reefs is only similar to the one observed in the Galicia Bank (7.1 ‰ 34 ) where the authors could not define a conclusive food source for the CWCs, suggesting a mixture of different sources 34 . In fact, isotopic enrichment might differ between deep-sea environments and conditions 35,44,70 depending on the available food sources, the metabolic rate of organisms and the isotopic composition of the surrounding water column. Furthermore, the observed δ 15 N enrichment in our study could also point to the presence of an additional trophic level between the phytodetritus and the corals, missed in our sampling. This gap could indicate that, in addition to fresh phytodetritus, zooplankton might be an important food source for the CWCs offshore Angola, as previously observed in other CWC reefs dominated by scleractinians 31,34,89,90 . Indeed, zooplankton is a nutritional rich food source for cold-water scleractinians 45,91 , octocorals 92,93 , and antipatharians 31,33,94 . The Angolan basin harbors abundant and diverse zooplankton communities 95 whose dial vertical migrations coincide with the water depth where CWCs are found (331 to 473 m) 96 . The zooplankton δ 15 N ratios (ranging from 3.5 to 10 ‰) from other regions of the Atlantic sampled in the same depth range (300 to 500 m) as our study 34,37,97-99 fit with the expected enrichment of the intermediate trophic level. Thus, zooplankton prey should be considered available to the examined CWCs. Accordingly, we hypothesize that the corals' diet is most likely based on fresh phytodetritus, when available 100 , but zooplankton might contribute the most to the CWCs diet. However, the role of zooplankton as primary food source in the Angola CWC reefs still needs to be investigated in future studies.
Black coral and octocorals occupy the same trophic guild of D. pertusum and M. oculata (cluster 2, Fig. 1) however, their δ 13 C and δ 15 N signatures are more enriched than those of the latter two. These differences might be explained by taxon-specific (morphological and physiological) requirements 36,101 , since colony morphology 102 and polyp size 103 influence the rates of food capture 104 and the selection of prey type and size 61 between CWC species. This possibly leads to a less-opportunistic feeding strategy and food selection 61 focused in capturing more of one type of food source, potentially zooplankton, resulting in differences in their trophic strategies 93 and, consequently, isotopic signatures.
The crustacean decapod Munida sp., a fish of the family Myctophidae, and a polynoid polychaete also belong to the same trophic group as CWCs. This result highlights their suspension-feeding behavior and, most likely, all of them exploit the same food sources as the CWCs. Moreover, given that small fish are known to preferably feed on zooplankton 97 , this finding further supports the hypothesis of the importance of zooplankton as a food source to cluster 2. On the other hand, the polychaete E. norvegica and the analyzed echinoderms (Asteroids, Ophiuroids and Echinus sp.) are the most 13 C-enriched organisms (δ 13 C from − 15.39 to − 12.81 ‰) with a corresponding mean TP of 4.16 ± 0.47 (cluster 3). The polychaete E. norvegica is known to live in close association with D. pertusum and M. oculata 13,[105][106][107] and to contribute to reef formation 106 , where the polychaete could benefit from extra food supply from the coral host by feeding directly on detritus in the corallites 105 . In the Angolan CWC reefs, E. norvegica was present at the basal part of the reef-building coral colonies 29 . The δ 15 N of E. norvegica is in the same range as the one for the other polychaete analyzed in this study. However, the comparative δ 13 C enrichment of E. norvegica indicates an exploitation of different food sources that could be related to its association with D. pertusum since it has been shown that, in the presence of the coral, E. norvegica assimilates more carbon through selective feeding on bigger particles 108 . It is possible that bigger particles consumed by E. norvegica indicate fresher particles, with a higher content of chlorophyll-a, resulting in 13 C-enrichment 109 .
The high standard deviation in δ 15 N of asteroids and in the δ 13 C of Echinus sp. could be attributed to the opportunistic feeding behavior of the organisms in these groups [110][111][112][113] . Given that all the organisms included in this trophic group are mobile species, they are potentially able to exploit different food sources within the reef, including detritus 31,114 , zooplankton 37,115 or, by directly predating corals and sponges 116 . The latter seems more likely, since ROV video observations have shown the sea urchin Echinus sp. on top of D. pertusum colonies (Fig. 3), and the dissection of collected specimens confirmed the presence of fragments of D. pertusum in their gut's contents 29 , supporting the evidence of its predatory feeding behavior. Moreover, echinoderms in the CWC reef presented the most enriched δ 13 C ratios (an enrichment of 5 ‰ compared to cluster 2). One possible explanation to the 13 C-enrichement of this group could be a "deep-sea sponge loop" 40, 70,117,118 , where the sponges' feeding on bacteria and DOM, will result in the release of 13 C-enriched particulate detritus that can be assimilated by associated fauna, both through detrital feeding and by directly feeding on sponges through a predatory pathway 40, 118 . However, this interpretation should be taken with caution since in situ experiments are needed to support a "deep-sea sponge loop".
Sponges are active filter-feeders and, therefore, it was expected that sponges act as primary consumers on the Angolan CWC reefs. However, sponges exhibited the highest δ 15 118,[122][123][124][125] , or even small zooplankton through the specialization of a group of sponges (Family Cladorhizidae) to utilize carnivory feeding modes 126,127 . In fact, the enriched δ 15 N values of hexactinellid sponges in the Angolan reefs are not entirely surprising since similarly high ratios have also been reported for deep-sea sponges in several other studies in the North Atlantic 37,79,98 , Pacific 125,128 , Arctic 70,114,129 and Antarctic 66,130 Oceans. The 15 N-enriched values of sponges could be attributed to intense nitrogen cycling pathways in the sponge's tissue 70,131 driven by (1) the sponge holobiont 37,79 , defined here as the sponge and its associated microbial communities, and/or (2) the sponge's feeding on heterotrophic re-suspended bacteria that are 15 N-enriched because of the uptake of waste material of higher trophic levels 70,125,130 .
The Angolan CWC reefs occur in the center of the local OMZ and OMZs are zones with thriving microbial communities 55 and complex nitrogen cycling dynamics 54,132,133 . The tissue of sponges is rich in microbial communities 53,129,134,135 and the nitrogen isotope fractionation in sponges changes according to their bacterial richness 53,70,125 , i.e., if High Microbial Abundance (HMA) sponges or Low Microbial Abundance (LMA) sponges 70,136 . The sponge holobiont takes energy from DOM 137 and, as a result of OM degradation, will release ammonium (NH 4 + ) 70,123,138 , an isotopically depleted metabolic end-product 139 , leading to increased δ 15 N in the sponge tissue. Moreover, symbiotic relationships with bacteria, capable of both denitrification and anammox 131,140,141 , play an important role in sponge survival under low oxygen environmental conditions 52,53,142,143 . Likewise, it has been shown by Middelburg et al. 52 that denitrification can occur in D. pertusum, due to the dominance of denitrifying bacteria in the coral's holobiont and this process is enhanced by low-oxygen conditions. It is beyond the scope of our study to identify the exact physiological mechanisms used by benthic organisms to cope with low-oxygen environments, but the observed δ 15 N ratios of sponges in our study hints that interactions with microbes could be a plausible mechanism to withstand the hypoxic conditions. Given the slightly enriched δ 15 N values of D. pertusum, the CWC holobiont could also potentially play a role in the coral's coping with the hypoxia conditions of the Angolan CWC reefs 50,52 , although this is more likely to be more significant for sponges given their enriched stable nitrogen isotope values.
This study gives the first insights into the trophic structure of the Angolan CWC reefs, where different feeding strategies are utilized by the organisms inhabiting in this OMZ. Our observations provide the first evidence pointing to an important role of zooplankton as food source for CWCs in Angola, and to a strong reliance of sponges, through their holobiont, on bacteria as food source in hypoxic conditions. The use of other biomarkers in future studies, such as compound specific stable isotope and fatty acids analyses, or in situ experiments with isotopically labelled food will contribute to a better understanding of CWCs and sponge's nutrition. We further suggest that, in the future, special attention should be given to the potential role of the holobiont in allowing CWCs and sponges to withstand the conditions of the Angolan and other OMZs.

Methods
Study area. The Angola basin is located along the SW African continent in the SE Atlantic Ocean and belongs to the Benguela Current Large Marine Ecosystem (BCLME) 144 . The region is influenced by the Benguela upwelling system, one of the world's most productive 145 , with an estimated primary production of 156 million www.nature.com/scientificreports/ t C yr -1146 . Induced by coastal parallel winds, the upwelling of nutrient-rich cold waters results in enhanced primary productivity 81 , while the remineralization of high concentrations of OM in the water column results in severe mid-depth oxygen depletion and in a pronounced OMZ 147 that coincides with the presence of the Angola CWC mounds 25 . In addition, the area receives a high input of riverine discharges from the Congo and Cuanza Rivers, which leads to further increase in primary production due to a high terrigenous nutrient input 11,82 . At approximately 17°S, the cold nutrient-rich waters transported northward by the Benguela Current interact with the warm nutrient-poor Angola Current 148 , forming the Angola-Benguela Frontal Zone (ABFZ) 149 . Oxygendepleted and nutrient-rich waters are characteristic from 70 to 600 m water depth, indicating the presence of the South Atlantic Central Water (SACW) 150 , which is transported southward by the Angola Current. The Angola CWC reefs cover long ridges to individual coral mounds that can reach up to 100 m above the surrounding sea floor. These CWC ridges and mounds have formed on millennial and longer time scales 151 , through successive periods of CWC reef development. They are composed of CWC fragments and other shells loosely embedded in fine hemipelagic sediments 25,29,30 . Sample collection. Samples were collected in January 2016 during the M122 ("ANNA") expedition on board the R/V Meteor 29 . In total, 18 reef sites topping seven different CWC mound complexes (Fig. 4, Table 2) were sampled for stable isotope analyses. Samples of organisms belonging to the taxa Porifera, Cnidaria, Arthropoda, Annelida, Echinodermata and Chordata were collected by means of a box corer (box dimensions: 50 × 50 cm, 55 cm high), a Van-Veen grab sampler or the Remotely Operated Vehicle (ROV) SQUID (MARUM, Bremen, Germany) 29 (Supplementary Table S1). Each organism was identified to the lowest possible taxonomic level, rinsed in seawater and put in plastic bags or vials.
To investigate potential food sources for the megabenthic fauna, three types of POM were collected: SPOM from the water column, settling material from the sediment trap (SPOM trap), and sediment. SPOM was collected at two depths (342 and 532 m) at 40 cm above the seafloor using a McLane phytoplankton pump, attached to the ALBEX lander (NIOZ), with a maximum of 7.5 L of water pumped every 2 h for a period of 48 h over 24 GF/F filters (47 mm Whatman™ GF/F filters pre-combusted at 450 °C) (for more details see Hanz et al. 11 ). Settling SPOM (SPOM trap) was also collected with a sediment trap (Technicap PPS4/3) attached to the ALBEX lander, with the collector at 2 m above the bottom over a sampling duration of 2.5 days, at both 342 and 526 m depth. Additionally, two sediment samples were collected at two depths (259 and 345 m): one with the box corer and another one with the Van-Veen grab sampler. All organisms and POM samples were stored on board at -20 °C and, afterwards, were freeze-dried in the laboratory and stored at − 20 °C until further analysis.

Sample preparation for stable isotope analyses.
Fragments of freeze-dried faunal samples were ground to powder with a mortar and, depending on the quantity of sample available, two subsamples of 10 to 50 mg were weighted. One subsample was left untreated for δ 15 N determination, whereas the other was acidified with the addition of 10% HCl drop-by-drop until effervescence ceased and used for δ 13 C determination. The www.nature.com/scientificreports/ acidification of samples is a common practice in stable isotope analyses and aims to remove inorganic calcium carbonate from the organisms, known to interfere with δ 13 C ratios 112 . All samples analyzed for δ 13 C were acidified, except fish samples due to their low carbonate content 152 . After acidification, the subsamples were ovendried at 50 °C for 72 h. Afterwards, three pseudo replicates of each subsample were weighted with a precision balance (± 0.001 mg) into tin capsules (11 × 4 mm, Elementar Microanalysis) to conduct the isotopic composition analyses. Depending on the taxonomic group, subsamples of 0.4 to 4 mg were weighted. Sample preparation for the determination of δ 15 N and δ 13 C for the three types of POM considered (filtered SPOM, SPOM trap and sediment) is described in detail in Hanz et al. 9 . These samples were acidified by fuming HCl (20%) vapor over night for δ 13 C determination. Three replicates of sediment (1.6 to 3.6 mg for δ 13 C and 9 mg for δ 15 N) and sediment trap material (1 mg δ 13 C and 4 to 5 mg for δ 15 N) were weighted into tin capsules. For SPOM, a ¼ section of each GF/F filter was transferred into tin capsules for δ 13 C and δ 15 N analyses.
The δ 15 N and δ 13 C values of filtered SPOM, SPOM trap and sediment were analyzed by a Delta V Advantage IR-MS coupled online to an elemental analyzer (Flash 2000 EA-IRMS) by a ConFlo IV (Thermo Fisher Scientific Inc.). Benzoic acid and acetanilide were used as standards for δ 13 C, whereas acetanilide, urea and casein were used as standards for δ 15 N (for more details see Hanz et al. 9 ).
Vienna Pee Dee belemnite (V.P.D.B.) for carbon, and atmospheric N 2 (Air) for nitrogen, were used as reference materials, and stable isotope values are here reported with respect to those. Precision is based on the standard deviation between replicate analyses and was < 0.50 ‰ for δ 15 N and < 0.20 ‰ for δ 13 C for faunal samples, and < 0.15 ‰ for δ 15 N and δ 13 C for POM.
Stable isotope ratios (δ 13 C and δ 15 N), in relation to the standards, were calculated as following: where R corresponds to 13 C/ 12 C or 15 N/ 14 N of the analyzed sample (R sample ) and standard used (R standard ).
Data analysis. The Trophic Position (TP) of consumers was calculated following Post 72 , as in similar studies on deep-sea habitats 98,115,153,154 . The TP of each consumer was calculated using the following equation: (2) TP n = + δ 15 N n − δ 15 N baseline /TEF Table 2. Metainformation of the number of samples collected (n), type of sample (Fauna, Sediment or suspended particulate organic matter (SPOM)) and sampling gear at the different sampling stations (Station ID). www.nature.com/scientificreports/ where TP n corresponds to the trophic position of the organism, δ 15 N n corresponds to its stable nitrogen isotope ratios, δ 15 N baseline represents the nitrogen isotopic composition of the baseline, TEF corresponds to the Trophic Enrichment Factor for δ 15 N and λ represents the trophic level occupied by the baseline. The δ 15 N of the baseline considered was 4.23 ± 1.65 ‰, corresponding to the mean ratio measured for SPOM (λ = 1). A TEF of 3.4 ‰ for δ 15 N was considered, since it has been regarded as appropriate in other CWC habitats 31,37 .
To explore the different trophic guilds of the Angolan CWC reefs, a k-means clustering analysis was applied to the δ 13 C and δ 15 N data, computed using the "factoextra" package 155 within R Studio 156 under R version 4.1.3 157 . The optimal number of clusters (k = 4) was investigated using the "NbClust" R package 158 by applying three different methods: elbow, silhouette, and gap statistic methods (Supplementary Fig. S2). The Pearson correlation between δ 13 C and δ 15 N ratios was calculated using the "cor.test" function available in the "stats" package in R. Since fauna and POM samples were collected along different CWC mounds, it was not possible in our study to assess how the stable isotopic ratios would differ between mounds.

Data availability
Dataset with samples metadata and carbon and nitrogen stable isotope values are available in Pangaea 159